Ontogenetic shift and feeding habits of the European hake (Merluccius merluccius L., 1758) in Central and Southern Tyrrhenian Sea (Western Mediterranean Sea): A comparison between past and present data

Abstract The present paper aims to investigate the ecological role of Merluccius merluccius, Linnaeus, 1758, in southern and central Tyrrhenian Sea (GSA 10, Resolution GFCM/33/2009/2 General Fisheries Commission for the Mediterranean), analyzing ontogenetic diet shifts, geographical variations on prey composition, and feeding habits. A total of 734 hake specimens ranging in size between 6 cm and 73 cm (Total Length, TL) were collected in 2018. In order to evaluate ontogenetic shifts in prey composition, samples were divided into five size classes and for each class the quantitative feeding indices have been calculated. The statistical analysis, based on index of relative importance percentage (%IRI), resulted in three trophic groups. The most abundant prey found in the immature hake specimens (size class I) were the Euphausiids, Stylocheiron longicorne and Mysidacea, while for samples with a total length over 10.5 cm were crustaceans and fish. Engraulis encrasicolus was the most abundant fish prey identified, followed by Boops boops and Myctophids. The high presence of Euphausiids, Mysids, Myctophidae, and Sternoptychidae in classes I, II, II, and IV (6–23 cm) showed the relevant role of mesopelagic fauna in hake diets, with an essential organic matter and energy flow from the mesopelagic to the epipelagic environment. Additionally, decapod crustaceans were found in the stomach contents of hakes belonging to class V (with size over 36 cm TL), which is notable considering that our study area includes an important decapod crustacean fishing area.


| INTRODUC TI ON
Mediterranean Sea is one of the most exploited ecosystems in the world, with a very high anthropogenic impact related to fisheries activities. According to Colloca et al. (2013), based on Scientific, Technical, and Economic Committee for Fisheries (STECF) data from 2010 (Carvalho & Doerner, 2014), 84% of all Mediterranean stocks were overexploited, with an increase up to 2014 (88%), declining to 75% in 2018 (FAO, 2020). Overexploited stocks include all existing demersal fish stocks, and seven crustaceans stocks out of nine, while a sustainable exploitation level was only showed by small pelagic stocks  (FAO, 2020). Indeed, the Southern Tyrrhenian Sea is an heavily exploited area, with a large trawling fleet composed by 2800 vessels (the third one for vessels number among the Italian fleets), contributing to 12% of total national landings (IREPA, 2011), and a peculiar heterogeneity of fisheries activities, with an high developed artisanal fisheries characterized by a large variability of fishing methods and target species (Cataudella & Spagnolo, 2011;Giordano, 1999;Perdichizzi et al., 2022) and the presence of one fishery exclusion zone which has been subject to a trawling ban since 1990 (Mangano et al., 2015;Pipitone et al., 2000;Rinelli et al., 2004). Therefore, it is important to monitor commercial fishing and conservation status of the mainly exploited stocks, such as M. merluccius.
It is essential to fully understand the ecology of several species, especially the most over exploited ones, to improve the multispecies approach of ecosystem-based fishery management and deepen the knowledge on ecological relationships within the marine trophic chains (D'Iglio, Albano, Tiralongo, et al., 2021;D'Iglio et al., 2022;Möllmann et al., 2014;Pikitch et al., 2004;Savoca et al., 2020;Stagioni et al., 2011;Zhou et al., 2010). Trophic network and ecological dynamics involving target species in the marine ecosystem can be clarified by a careful study and description of their diet and feeding habits (Angelini et al., 2016;Chipps & Garvey, 2006;Punt et al., 2016;Riccioni et al., 2018). These studies should be increased to prevent a fishing-induced decline of marine food web trophic levels (Shackell et al., 2010) setting also a conscious exploitation across the trophic levels .
Prey-predator relationships, and their changes in time, are the basis of multispecies population dynamics and complex ecosystem models (Carrozzi et al., 2019). The abundance decrease of piscivorous predators in Mediterranean Sea related to fisheries overexploitation (Colloca et al., 2013) is a serious problem for marine ecosystems, with poorly understood consequences for entire marine food chains (Leroux & Loreau, 2015;Lotze et al., 2011).
The European hake (Merluccius merluccius, Linnaeus, 1758) is an essential predator of the deeper shelf-upper slope Mediterranean communities. This species inhabits a wide depth range (20-1000 m) throughout the Mediterranean Sea and the north-eastern Atlantic regions (Carpentieri et al., 2005;Fischer, 1987) and it is considered a nektobenthic species. A size-based bathymetric segregation can usually be observed in hake populations, with larger individuals living in waters deeper than 200 m, while juveniles mainly inhabit the coastal shelf (Meiners, 2007). The European hake is an opportunistic predator since its feeding habits show geographical differences in prey richness and availability (Bozzano et al., 2005;Cartes, Hidalgo, et al., 2009;Cartes, Maynou, et al., 2009;Carrozzi et al., 2019;Papaconstantinou & Stergiou, 1995;Velasco & Olaso, 1998). Maybe, this great variety of feeding habits is due to its wide bathymetric distribution (Carpentieri et al., 2005;Cartes, Hidalgo, et al., 2009;Cartes, Maynou, et al., 2009;Cartes et al., 2004). Its opportunistic feeding behavior could also induce specimens to feed on trawl gears discard, represented by organisms killed or injured by this fishing activity, as reported by previous studies on other demersal predator species Kaiser & Spencer, 1994).
The M. merluccius specimens show changes in diet composition and feeding habits related to their ontogenetic development.
Planktonic species, such as euphausiids and mysids, are mainly preyed by immature and juvenile individuals (total length >15 cm, indicated as size classes I and II in the present paper). These follow the preys (mainly zooplanktonic species as Euphausiacea) during their nictemeral vertical migration, moving upward and downward through the water column (Orsi Relini et al., 1997). Small fishes, cephalopods, and nektobenthic decapods are mainly preyed by hake individuals with a total length from 10.5 to 20 cm, (indicated as size classes II and III in the present paper). Finally, larger pelagic and nektobenthic fishes are the typical preys of mature hakes, up to 20 cm of total length (size classes IV and V of present paper).
Although many authors deal with this topic through all the Mediterranean areas, there are only two studies focused on
The aim of the present study was to investigate, in relation to the size class, the changes in the diet composition and in the feeding habits of the species Merluccius merluccius from the central and southern Tyrrhenian Sea. Monitoring trophic variations in overexploited species of high commercial value is essential for stocks and demersal community conservation, improving fisheries management, especially in a high exploited area, such as the Southern Tyrrhenian Sea.

| Study area
The investigated area extends in southern and central Tyrrhenian Sea (GSA10) (Figure 1), from the Garigliano river mouth, on the Lazio border, to Capo S. Vito, the westernmost part of northern Sicily, covering a 20,225 km 2 area, with a bathymetric depth between 10 and 800 m. Its coastal extension is 1129 km, involving five regions: Lazio, Basilicata, Campania, Calabria, and Sicily. GSA10 is heavily exploited by a large trawling fleet. Moreover, it is characterized by one fishery exclusion zone which has been subject to a trawling ban since 1990 (Pipitone et al., 2000;Rinelli et al., 2004) and a region typically oligotrophic, in the southern part (Povero et al., 1990).

| Stomach data
A total of 734 European hakes, M. merluccius, individuals ranging from 6 and 73 cm total length (TL) were collected from the GSA10 during Summer (by MEDITS 2018 bottom-trawl survey) and during the entire year by commercial landings of the fishing fleets (CAMPBIOL 2018 survey). The collected samples were frozen on board to prevent digestion of stomach contents and taken to the laboratory, where each specimen was measured (total length, TL cm), weighed (total weight, TW g), and sexed with the relative degree of sexual maturity (Follesa & Carbonara, 2019).
Stomach repletion status was detected using a five-stage macroscopic scale (1 empty; 2 full <50%; 3 full >50%; 4 bursting; 5 everted, stomach inside the mouth). The stomachs were preserved in ethanol 70% + glycerin 5%. Each prey item was identified to the lowest possible taxonomic level, counted and weighed; for each one (species or major Taxa) was detected the digestion degree (1 = undamaged; 2 = almost digested; 3 = highly digested). The anatomical undigested parts, as mouth parts, entire heads capsules, otoliths, eyes, telsons, carapace, were used to count any prey. The double-counting was avoided by considering only one kind of anatomical remains for each preys group, depending on their presence within the stomach contents (e.g., only carapace or telsons, only eyes or otoliths, only heads capsules or fishes' columns).
Prey items in a state of advanced digestion were grouped into undetermined fish, crustaceans, and cephalopods. The vacuity index (VC), obtained by VC = (N e /N) × 100, where Ne is the number of empty stomachs and N is the number of total stomachs, was calculated; in the VC we did not consider the everted stomachs. To evaluate the contribution of each prey items, the following parameters were calculated: the percentage of abundance composition (%N), the percentage of biomass composition (%W), and the frequency of occurrence (%F) (Hyslop, 1980). These indices are necessary to calculate the Relative Importance Index, IRI = %F (%N + %P), expressed as (Cortés, 1997). To evaluate the diet variations related to growth, five-length classes were assessed using an incremental scale by 5 cm for length classes I, II, and III. The first class (I) included all immature specimens (<10 cm). The second class (II) included specimens with a total length between 10.5 and 15 cm, while to the third class (III) belonged individuals with a total length between 15.5 and 20 cm. The largest individuals (>20.5 cm) were grouped into two heterogeneous classes: IV (20.5-32.5 cm of total length) and V (total length >32.5 cm). Univariate and multivariate statistical analyses were performed by using Past (V. 4) and PRIMER6-E. p value was set at p < .05.

| RE SULTS
As shown in Table 1, the vacuity index (VC) was increased in value from size classes I to II, decreasing in the other size classes.
Concerning the everted stomachs number, size class IV showed the highest value, followed by size class III, with the lowest value showed by size class V.
The content analysis of 274 sampled stomachs showed a total of 120 preys, which were found to belong to 28 taxa. The overall analysis of the diet, based on the %IRI, showed that fishes were the preferred prey (% IRI = 95.29; %F = 80.5), followed by Mysids (%IRI = 2.57) and Euphausiids (% IRI = 1.25) ( Table 2).
The Euphausiids and Mysidacea were the most relevant (higher IRI values) prey for immature hake (size class I < 10 cm TL), while decapoda and fish were the preferred prey for hake larger than 10.5 cm of TL (size classes II, III, IV, and V) (  (Table 2) and abundant preys (higher %N) ( Table 2).
Cluster analysis and MDS ordination grouped the whole set of data, by maturity classes, into two main clusters. Cluster I included only the specimens belonging to Class I, while the second one included the specimens belonging to the Classes II, III, IV, and V with similar values of %IRI (Figure 2). In detail, classes III, IV, and V showed the 60% of similarity.
One-way ANOVA analysis was performed to assess dietary composition differences between maturity stage of the M. merluccius specimens (p < .05). Indeed, class I was different from all other classes (p < .05), while classes II, III, IV, and V did not differ significantly in their diets (p > .05).
The ANOVA confirmed the similarity of diet between maturity stages, which was driven by the large contributions of Euphausiacea and Osteichthyes as shown by PCoA, explaining 42% of total variation on the first axis ( Figure 3).

| DISCUSS ION
The present paper provides, for the first time, an analysis of the diet and feeding habits of the Merluccius merluccius specimens from the entire area of the central and southern Tyrrhenian Sea (GSA10).
Our findings enrich and implement the data already reported by Modica et al. (2015) and Sinopoli (2012), for the southern part of and characterized by a low incidence of cephalopods. Size-related dissimilarities in diet composition could be related to genetic needs and/or differences in spatial distribution (Flamigni, 1983;Jukic & Arneri, 1984;Velasco & Olaso, 1998). The high density of juveniles belonging to different species, included European hake, is sustained by these habitats, thanks to zooplankton and micronekton aggregations enhanced by primary production increase and convergence processes (Cartes, Hidalgo, et al., 2009;Cartes, Maynou, et al., 2009;Colloca et al., 2004;Fiorentino et al., 2003;Garofalo et al., 2011).
As also shown by the results obtained in this study, the pres-  Layer") during their nictemeral migration toward superficial and shallower waters. This is a clear example of inverse energy flow from deeper to epipelagic waters, essential for all demersal species. Since Myctophidae and Sternoptychidae live in depths greater than 600 m (D'Onghia et al., 2004) and hake, especially juveniles and small adults, are generally distributed above 330 m in the studied area (Biagi et al., 2002), we could assert that this area is characterized by an inverse energy flow from deeper to epipelagic water, probably reallocated along the neritic food chain. The upward transfers may be increased by the consumption of mysids and euphausiids in TA B L E 2 Diet composition of the Merluccius merluccius specimens collected from southern and central Tyrrhenian Sea (GSA 10) with %F (frequency of occurrence), %W (percentage in biomass), %N (percentage of number) IRI (index of relative importance) and %IRI (index of relative importance expressed as percentage) values for each prey item Sicily, these species are mainly preyed by smaller juvenile hakes (Carrozzi et al., 2019;Fanelli et al., 2018), with E. encrasicolus as the most abundant prey for hake below 14 cm of TL (Classes I and II of present paper), and larger hake which mainly prey on larger bony fishes (T. trachurus, Lepidopus caudatus, Euphrasen, 1788) and decapods (Carrozzi et al., 2019). The abundance of preys belonging to the Engraulidae and Clupeidae families in stomach contents of the hake analyzed in the present paper is probably due to the ecological features of the Tyrrhenian Sea, especially in the central region (Bauchot, 1987). In this area, especially during winter and autumn, they are the most available and abundant preys, largely distributed on continental coastal shelf and forming schools usually deeper than 25 m.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.